The ingestion of pathogens, especially bacterial pathogens, including viruses and other disease-causing microorganisms, is a common problem in most animals. It is well known that pathogens cause diseases in animals, with numerous harmful effects including weight loss, diarrhea, abdominal obstruction, and renal failure. In the case of immunosuppressed or underfed animals, the effects of diarrhea may be even fatal. Pathogens are often transferred among animals under poor hygienic conditions, and even when suitable care is available, contagion may not be avoided.
Extreme health risks result when humans consume pathogens in contaminated food products such as sprouts, lettuce, meat products, unpasteurized milk or juice, water contaminated with sewage waters, etc. The problem is particularly frequent in beef and the dairy sector. Pathogens present in the udder of cows or in the milking equipment may be a source of contamination for raw milk. Beef may be contaminated in the slaughterhouse, and pathogen organisms may be subsequently mixed with large amounts of meat during grinding. Serious and life-threatening infections may occur when humans eat meat, especially ground beef, which has not been cooked enough so as to kill any pathogen present in the beef. This problem is difficult to solve because contaminated meat often looks and smells perfectly normal. Further, the number of pathogenic organisms necessary to cause a disease condition is extremely small, making detection very difficult. Pathogens that cause diseases in the intestinal zone are known as enteropathogens. Examples of these bacteria include Staphylococcus aureus, specific strains of Escherichia coli (E. coli), and Salmonella spp. While most of the hundreds of E. coli strains are harmless and live in the intestines of animals, including humans, some strains such as E. coli 0157:H7, 0111:H8, and 0104:H21, produce great amounts of powerful toxins closely related or identical to the toxin produced by Shigella dysenterieae. These toxins may cause severe pain in the small intestine, often producing harmful effects in the intestinal zone and in extreme cases, diarrhea. E. coli 0157:H7 and other enterohemorrhagic strains may also cause acute hemorrhagic diarrhea, characterized by severe abdominal obstruction and abdominal bleeding. In children, this may develop into a rare but fatal disorder called hemolytic uremic syndrome (“HUS”), characterized by renal failure and hemolytic anemia. In adults, it may develop into a condition known as thrombotic thrombocytopenic purpura (“TTP”), which involves HUS in addition to fever and neurological symptoms, and may have a mortality rate as high as of 50% in the elderly.
A reduction in the risk of diseases caused by food-borne pathogens may be achieved by controlling potential contamination sources. The beef industry has recognized the need of increasing the control of pathogens prior to harvest, particularly the control of E. coli 0157:H7 and other hemorrhagic serotypes, to avoid contamination of products, potential contact with humans, and transmission of pathogens during meat processing. In particular, raw or undercooked hamburgers (ground beef) have been involved in many outbursts or documented epidemics as containing E. coli 0157:H7 and other hemorrhagic serotypes.
Thus, there persists a recognized need for providing compositions and methods for reducing or eliminating the growth of enteropathogens such as E. coli 0157:H7 and other hemorrhagic serotypes, for the benefit of human and animal health.
Therefore, for the benefit of consumers, there is an important need of reducing or eliminating the growth of enteropathogens in animal meat and milk before their harvest. Such reduction or elimination of pathogens in animals intended consumption will provide a better protection for beef consumers, in dairy, and other food products against the risk of consuming said pathogens.
A very common solution to this problem has been the provision of antibiotics to the animals; however, this solution is not only costly, but may also lead to the generation or selection of antibiotic-resistant bacterial strains. Also, as is known, the treatment with antibiotics, in particular oral antibiotics, may modify or destroy the gastrointestinal flora. These antibiotics may exert a negative effect on the general health. Said negative or undesirable effect, consists in partly destroying the healthy bacteria naturally living in the body. For example, in the intestine there are healthy bacteria that usually live there: biphidobacteria and lactobacilli, that are part of the intestinal flora. Said intestinal microbiota constitute a natural defense for protection against stomach and intestine infections; infections which finally will cause problems such as diarrhea.
On numerous occasions, patients on antibiotics have diarrhea, due to destruction of the bacteria that naturally live in the intestine, and protect them against infections.
Likewise, women on antibiotics for a bacterial infection may suffer from mycosis (fungi) at vaginal level, as the antibiotics also kill the bacteria acting as natural defense (lactobacilli).
It has thus been shown that the gastrointestinal microbiota play a number of vital roles in maintaining normal function of the gastrointestinal tract and overall physiological health. For some experts, the key to good health resides in the intestine, whose role in the human body has been compared to that played by the roots of a tree. And, in fact, the intestine is not just an absorption organ. It is the most relevant site of action of the immune system and of non-specific protective mechanisms, as it is precisely in the intestines where they are most active. Its immunocompetent cells recognize pathogenic agents and activate the production of T lymphocytes that, in turn, differentiate into plasma cells and segregate non-specific antibodies.
When we are born, the gastrointestinal tract is sterile but shortly after a complex set of approximately 400 different types of microorganisms settles down permanently which work in harmony at maintaining the health. This microflora—the intestinal flora—weights over one kilogram, it may comprise up to 100 billions of different microorganisms which have an overall metabolic activity similar to that of a human liver. Once the microflora has settled, it may be negatively affected by factors such as consumption of very refined food poor in fibers, antibiotic treatments, and stress, among others.
For example, growth and metabolism of the many individual bacterial species living in the gastrointestinal tract depend mostly on the available substrates, mainly derived from the diet. See, for example. Gibson et al., 1995. Gastroenterology 106: 975-982; Christi, et al., 1992. Gut 33: 1234-1238; Gorbach, 1990. Ana. Med. 22: 37-41; Reid et al, 1990. Clin. Microbiol. Rev. 3: 335-344. These disclosures have led to different approaches intended to modify the structure and metabolic activity of the gastrointestinal tract through the diet, especially including probiotics, which are live microbial food supplements.
As it is known that pathogens live in many different areas of the digestive system of animals, it has been found beneficial to supply and/or reinforce the naturally-occurring organisms in these areas which are effective for inhibiting pathogenic growth throughout the digestive tract, such as in the rumen, small intestine, and large intestine.
Probiotics, when introduced into the gastrointestinal tract, may influence the gastrointestinal microflora and play a beneficial role in the human or animal host. The term “probiotic” derives from Greek “for life”. It was first used to describe substances secreted by microorganisms capable of stimulating the growth of other microorganisms (Lilly and Stillwell, 1965, Probiotics: growth-promoting factors produced by microorganisms. Science. February 12; 147:747-8).
In 1992, Havenaar suggested, as a definition for probiotics, “a viable mono- or mixed culture of microorganisms, which applied to animals or man, beneficially affects the host animal by improving the properties of the indigenous microflora” (Havenaar et al., 1992, Selection of strains for probiotic use. In: Probiotics, the Scientific Basis (Fuller R., ed.), pp. 209-224. Chapman and Hall, London, UK). Havenaar's definition was the first using the term probiotic for both humans and animals.
Taking into account the current applications and proven effects of probiotics, Salminen et al (1999), Probiotics: how should they be defined. Trends Food Sci. Tech.; 10:107-110), proposed a new definition: “probiotics” are preparations of microbial cells or components of microbial cells that exert a beneficial effect on health and comfort of the host. This definition includes microbial cells (viable or non-viable) and parts of cells as probiotics, but not metabolites such as antibiotics. This definition also indicates that the application of probiotics is not restricted to its use in food.
“Probiotics” are considered as viable microbial preparations that promote the health of an individual by preserving a healthier microflora in the intestine. A microbial preparation is commonly accepted as a probiotic when it as a known effect and mode of action. Probiotics bind to the intestinal mucosa, colonize the intestinal zone and also prevent settling of deleterious microorganisms into the intestine. An essential requirement for its action is that they should reach the intestinal mucosa in an appropriate and viable way without being destroyed at the upper part of the gastrointestinal tract, especially by influence of the low pH values prevailing in the stomach.
It is known that the low pH values in the stomach in addition to the antimicrobial action of pepsin provide an efficient barrier against the entry of bacteria into the intestinal zone. The pH of the stomach ranges from 2.5 to 3.5, but may reach values as low as pH 1.5, or as high as pH 6 or higher after food ingestion. The type of food affects stomach emptying. Normally, food remains in the stomach from two to four hours: however, liquids leave the stomach in about 20 minutes. Extensive in vitro tests have been used for selecting gastric tolerance, including lactic acid producing bacteria tolerant to acids (Charteris et al, 1998, Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J. Appl. Microbiol. May; 84(5):759-68; Clark et al, 1993, Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: III. Cult. Dairy Prod. J. 29:18-21; Chou and Weimer, 1999, Isolation and characterization of acid- and bile-tolerant isolates from strains of Lactobacillus acidophilus. J Dairy Sci. January; 82(1):23-31).
Another barrier that probiotic bacteria must overcome is the small intestine. The adverse conditions of the small intestine include the presence of bile salts and pancreatin. The transit time of food through the small intestine generally comprises between one and four hours. Lactic acid bacteria resistant to bile salts may be selected by assaying their survival capacity in the presence of bile salts and their growth on selective media with varying levels of bile (Gilliland et al, 1984, Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J Dairy Sci. December; 67(12):3045-51; Ibrahimand Bezkorovainy, 1993, Survival of bifidobacteria in the presence of bile salt. J Animal Sci. 62:351-354; Clark and Martin, 1994, Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: II—tolerance to simulated pH of humans stomach. Cult. Dairy Prod. J. 6:11-14; Chung et al, 1999, Screening and selection of acid and bile resistant bifidobacteria. Int. J. Food Microbiol. 47:25-32). A concentration of 0.15-0.3% of bile salts is a suitable concentration for selecting probiotics for human use.
After surviving the passage through the upper gastrointestinal tract, probiotic bacteria need to attach to the intestinal epithelium in order to colonize and remain in the gastrointestinal tract. The complexity of the intestinal mucosa and its microflora make it very difficult to study bacterial attachment in vivo.
Live probiotic microorganisms may provide advantages either during the preparation of fermented probiotic food or in the digestive tract of the host. After fermentation, texture and flavor of the raw materials are perceptibly improved; harmful effects of some feed components may be reduced, for example food intolerance and allergies caused by certain oligosaccharides and proteins; levels of amino acids and vitamins may be improved, which enhances the nutritional value of food; and sugars and other components that promote food decay may be removed, leading to a longer potlife and improving the safety of food products. Also, there is evidence that the bioavailability of calcium, zinc, iron, manganese, copper, and phosphorus is greater in fermented yoghurt as compared to milk. Studies have also showed an increase in riboflavin and niacin in yoghurt, vitamin B6 Cheddar cheese, vitamin B12 in quark and folic acid in a variety of products including yoghurt, quark, Cheddar cheese, and sour cream. Enzymatic hydrolysis of probiotic microorganisms has also demonstrated to enhance bioavailability of proteins and fats. Bacterial protease may increase the production free amino acids that may benefit the nutritional condition of the host, especially if said host has an endogenous protease deficiency.
In the food manufacturing industry, the lactic bacteria used as protecting cultures must have the ability to adapt themselves to the prevailing conditions in the corresponding product and must also show competitive ability. In most beef products, lactic bacteria must tolerate relatively high salt concentrations, and should be able to develop in the presence of nitrite at relatively low temperatures. Biological preservation of food is an important alternative to preservation with non-biodegradable chemical compounds which are toxic for humans.
It has also been disclosed that consumption of food containing viable probiotics produces health benefits including (1) alleviation of intestinal disorders such as constipation and diarrhea caused by an infection by pathogenic organisms, antibiotics, or chemotherapy; (2) stimulation and modulation of the immune system; (3) anti-tumoral effects resulting from inactivation or inhibition of carcinogenic compounds present in the gastrointestinal tract by reduction of intestinal bacterial enzymatic activities such as O-glucuronidase, azoreductase, and nitroreductase; (4) reduced production of toxic final products such as ammonia, phenols and other protein metabolites known to influence hepatic cirrhosis (5) reduction of serum cholesterol and arterial pressure; (6) maintenance of mucosal integrity; (7) alleviation of lactose intolerance symptoms; (8) prevention of vaginitis.
Examples of probiotic organisms include, but are not limited to, bacteria capable of growing, at least temporarily, inside the gastrointestinal tract, of displacing or of destroying pathogenic organisms, as well as providing additional advantages to the host.
It is known that certain bacteria, in particular bacteria isolated from healthy human or animal gastrointestinal tracts, as well as certain lactic acid bacteria such as Lactobacillus, have a prophylactic and therapeutic effect in gastrointestinal diseases, such as gastrointestinal infections. For this purpose, the administration of preparations containing these microorganisms (viable) to humans or animals is also known. After administration, these probiotic bacteria (also called eubiotic) compete with the pathogenic bacteria for food and/or binding sites on the gastrointestinal wall, such that their number is reduced and infections are thus reduced or prevented.
For many years, lactic acid bacteria have been used as fermenting agents for food preservation by reason of their low pH and the action of the fermentation products generated during their fermentation activity which inhibits the growth of harmful bacteria. To this end, lactic acid bacteria not well characterized yet have been used for preparing a variety of food products such as dry fermented meat products, cheese, and other fermented dairy products.
Recently, these lactic acid bacteria have attracted some attention because it has been found that some strains exhibit valuable characteristics for digestion in humans and animals. In particular, specific strains of the genera Lactobacillus or Biphidobacterium capable of colonizing the intestinal mucosa and assisting in the maintenance of wellbeing of humans and animals were found.
The best-known probiotics are lactic-acid generating bacteria (that is, lactobacilli and biphidobacteria), extensively used in yoghurts and in other milk products. These probiotic organisms are non-pathogenic and non-toxic, they maintain viability during storage, and survive the passage through the stomach and the small intestine. As the colonization of the host by probiotics is not permanent, they must be consumed on a regular basis in order to achieve persistent health-promoting characteristics. Commercial probiotic preparations generally comprise mixtures of lactobacilli and biphidobacteria, but species of yeast such as Saccharomyces have also been used.
In this aspect, several patent applications disclose specific strains of Biphidobacterium, Lactobacillus and, to a lesser extent, Enterococcus (E. faecium) and their beneficial effects on diarrhea, immuno-modulation, hypersensitive reactions or infections by pathogenic microorganisms.
In spite of the above-mentioned beneficial effects of these probiotics, as the bacteria that may be administered for treating gastrointestinal disorders also have preferred adhesion and/or growing sites in the gastrointestinal tract, and these sites may be different from the growing sites of the deleterious microorganisms to be controlled, administration of certain types of probiotic bacteria may not be of help against certain types of gastrointestinal infections.
Thus, Lactobacillus, Lactococcus, or Micrococcus may be located in the mouth, and preferably in the small intestine extending even to the ileum. Accordingly, administration of these bacterial genera will not be of great use against infections by pathogenic microorganisms, such as E. coli, Salmonella, Clostridium, Shigella, and Campylobacter, because they develop in other parts of the gastrointestinal tract, such as the colon.
On the other hand, biphidobacteria and enterococci grow in the anaerobic part of the gastrointestinal tract. Biphidobacteria are preferably located in the colon. Species of Enterococcus are preferably located in ileum and colon.
Another difficulty observed with many strains of enterococci useful as probiotics (for example, E. faecium), is that they show a natural resistance to the antibiotics of clinical use in man. Currently, the trend is not to incorporate bacteria with multiple resistance to antibiotics as additives of food products. There is a possibility of transferring said resistance to saprophytic bacteria colonizing the intestine. In addition, due to a condition of immunosupression (AIDS; chemotherapy in cancer, congenital immunologic dyscrasia, etc) or to an abdominal trauma, this antibiotic resistant probiotic bacteria that colonized the intestine may advance to the peritoneum or to the blood and originate infections difficult to treat due to their multi-resistance.
Accordingly, there persists a need for providing a therapeutic method as an alternative to prescription of highly efficient antibiotics and which would work in acute, as well as preventive, treatment scenarios with inhibitory activity against pathogenic bacteria, including those that are resistant to those antibiotics currently used for infections in humans and animals. In addition, the new agent should have inhibitory activity against other pathogenic organisms such as parasites and fungi.
There is also a need for this therapeutic method to be efficient against infections by pathogenic microorganisms not localized in those parts of the gastrointestinal tract where Biphidobacterium and Lactobacillus are localized, and that it also capable of being administered in combination with antibiotics.
An additional need is to provide a new probiotic strain which is not resistant to antibiotics of clinical use such as glycopeptides (vancomicin, teicoplanine), carbapenemes (impipenem, meropenem) and ampicillin and with a broad inhibitory spectrum against Gram positive and Gram negative bacteria, as well as against other parasitic, fungal and viral pathogens.